THE FORMATION OF BASAL BODIES (CENTRIOLES) IN THE RHESUS MONKEY OVIDUCT

Basal body replication during estrogen-driven ciliogenesis in the rhesus monkey (Macaca mulatta) oviduct has been studied by stereomicroscopy, rotation photography, and serial section analysis. Two pathways for basal body production are described: acentriolar basal body formation (major pathway) where procentrioles are generated from a spherical aggregate of fibers; and centriolar basal body formation, where procentrioles are generated by the diplosomal centrioles. In both pathways, the first step in procentriole formation is the arrangement of a fibrous granule precursor into an annulus. A cartwheel structure, present within the lumen of the annulus, is composed of a central cylinder with a core, spoke components, and anchor filaments. Tubule formation consists of an initiation and a growth phase. The A tubule of each triplet set first forms within the wall material of the annulus in juxtaposition to a spoke of the cartwheel. After all nine A tubules are initiated, B and C tubules begin to form. The initiation of all three tubules occurs sequentially around the procentriole. Simultaneous with tubule initiation is a nonsequential growth of each tubule. The tubules lengthen and the procentriole is complete when it is about 200 mµ long. The procentriole increases in length and diameter during its maturation into a basal body. The addition of a basal foot, nine alar sheets, and a rootlet completes the maturation process. Fibrous granules are also closely associated with the formation of these basal body accessory structures.     

A possible cause of termination of cell growth in the cellular slime mold Dictyostelium discoideum.

The gill ctenidium growth tip of the lamellibranch mollusc Aequipecten irradians recapitulates the temporal development of ciliated gill filaments and related structures in a spatial fashion. This “meristematic” relationship has allowed a study of basal body formation and ciliogenesis in adjacent cells of gill filament papillae at stages of progressively more advanced relative development. Basal bodies appear to originate quite rapidly, subsequent to the appearance of a complex of dense granules, quite reminiscent of the “condensation forms” or “procentriole precursors” typically seen in vertebrate ciliogenesis. Unlike basal body generation in higher forms, that in Aequipecten shows no obvious organized intermediate stages. During ciliation, randomly-oriented, nearly complete procentrioles are found concomitantly with actively-functioning basal bodies. Cilia formation in more advanced, already-ciliated cells is again preceded by the presence of granular complexes. Ciliogenesis in this mollusc thus shares with certain lower forms the property of very rapid basal body formation but, like many higher forms, it is preceded by the formation of a granular precursor complex, presumably consisting of particulate microtubule protein.     

The orientation of ciliary basal bodies in quail oviduct is related to the ciliary beating cycle commencement

In mature ciliated cells, the basal feet associated to the basal bodies point out in the direction of the effective stroke of the ciliary beating. In contrast, during ciliogenesis, the basal feet of the newly anchored basal bodies are randomly oriented. The reorientation of basal bodies occurs during the beginning of the coordinated beating cycle of the cilia.     

Immunocytochemical localization of myosin during ciliogenesis of quail oviduct

Myosin has been localized during ciliogenesis of quail oviduct by immunocytochemistry (immunofluorescence, immunoperoxidase, immunogold labeling) using a previously characterized monoclonal antibody. In ovariectomized quail oviduct many undifferentiated epithelial cells present a primary cilium arising from one of the diplosome centrioles. Myosin is associated with material located between the two centrioles. In contrast, in estrogen-stimulated quail oviduct, the material preceding the procentioles is never labeled. Basal bodies become labeled just before their migration toward the apical plasma membrane. During the anchoring phase, the labeling is mainly associated with the basal feet. In mature ciliated cells, myosin appears associated with an apical network embedding the basal bodies. This network is connected to a myosin-rich belt associated with the apical junctional complex which differentiates at the beginning of centriologenesis. The association of myosin with migrating basal bodies suggests that myosin could be involved in basal body movements.     

Two appendages homologous between basal bodies and centrioles are formed using distinct Odf2 domains

Ciliogenesis is regulated by context-dependent cellular cues, including some transduced through appendage-like structures on ciliary basal bodies called transition fibers and basal feet. However, the molecular basis for this regulation is not fully understood. The Odf2 gene product, ODF2/cenexin, is essential for both ciliogenesis and the formation of the distal and subdistal appendages on centrioles, which become basal bodies. We examined the effects of Odf2 deletion constructs on ciliogenesis in Odf2-knockout F9 cells. Electron microscopy revealed that ciliogenesis and transition fiber formation required the ODF2/cenexin fragment containing amino acids (aa) 188–806, whereas basal foot formation required aa 1–59 and 188–806. These sequences also formed distal and subdistal appendages, respectively, indicating that the centriole appendages are molecularly analogous to those on basal bodies. We used the differential formation of appendages by Odf2 deletion constructs to study the incorporation and function of molecules associated with each appendage type. We found that transition fibers and distal appendages were required for ciliogenesis and subdistal appendages stabilized the centrosomal microtubules.     

THE MORPHOGENESIS OF BASAL BODIES AND ACCESSORY STRUCTURES OF THE CORTEX OF THE CILIATED PROTOZOAN TETRAHYMENA PYRIFORMIS

Dividing cells of Tetrahymena pyriformis were observed by transmission electron microscopy for signs of morphogenesis of cortical structures. The earliest stage of basal body development observed was of a short cylinder of nine single tubules connected by an internal cartwheel structure. This is set perpendicular to the mature basal body at its anterior proximal surface under the transverse microtubules and next to the basal microtubules. Sequential stages show that the single tubules become triplet tubules and that the "probasal bodies" then elongate and tilt toward the organism's surface while maintaining a constant distance of 75–100 mµ with the "parent." The new basal body after it is fully extended contacts the pellicle, and then assumes a parallel orientation with and moves anterior to the parent basal body. The electron-opaque core in the lumen of the basal body and accessory structures around its outer proximal surface appear after the developing basal body has elongated. These accessory structures associating with their counterparts from other basal bodies and with the longitudinal microtubules may play a role in the final positioning of basal bodies and thus in the maintenance of cortical patterns. Observations on a second sequence of basal body formation suggest that the oral anlage arises by multiple duplication of somatic basal bodies.     

Centriole and basal body formation during ciliogenesis revisited.

This review is concerned with the formation during ciliogenesis of centrioles and basal bodies, primarily in epithelial multi-ciliated cells from the developing vertebrate respiratory and reproductive tracts. During ciliated cell differentiation, in these as well as in other cell types, cilium formation is preceded by the formation of centrioles assembled from precursor structures having little resemblance to the mature organelle. The origin, composition and function of the centriole precursor structures in generating large numbers of centrioles in a short period of time during ciliogenesis is discussed. This review also focuses on the biochemistry of centrioles and basal bodies and on recent experimental evidence that DNA might be associated with these structures.     

CENTRIOLE REPLICATION : A Study of Spermatogenesis in the Snail Viviparus

This paper describes the replication of centrioles during spermatogenesis in the Prosobranch snail, Viviparus malleatus Reeve. Sections for electron microscopy were cut from pieces of testis fixed in OsO₄ and embedded in the polyester resin Vestopal W. Two kinds of spermatocytes are present. These give rise to typical uniflagellate sperm carrying the haploid number of 9 chromosomes, and atypical multiflagellate sperm with only one chromosome. Two centrioles are present in the youngest typical spermatocyte. Each is a hollow cylinder about 160 mµ in diameter and 330 mµ long. The wall consists of 9 sets of triplet fibers arranged in a characteristic pattern. Sometime before pachytene an immature centriole, or procentriole as it will be called, appears next to each of the mature centrioles. The procentriole resembles a mature centriole in most respects except length: it is more annular than tubular. The daughter procentriole lies with its axis perpendicular to that of its parent. It presumably grows to full size during the late prophase, although the maturation stages have not been observed with the electron microscope. It is suggested that centrioles possess a constant polarization. The distal end forms the flagellum or other centriole products, while the proximal end represents the procentriole and is concerned with replication. The four centrioles of prophase (two parents and two daughters) are distributed by the two meiotic divisions to the four typical spermatids, in which they function as the basal bodies of the flagella. Atypical spermatocytes at first contain two normal centrioles. Each of these becomes surrounded by a cluster of procentrioles, which progressively elongate during the late prophase. After two aberrant meiotic divisions the centriole clusters give rise to the basal bodies of the multiflagellate sperm. These facts are discussed in the light of the theory, first proposed by Pollister, that the supernumerary centrioles in the atypical cells are derived from the centromeres of degenerating chromosomes.     

Basal body replication in green algae--when and where does it start?

Basal body duplication in the green alga Spermatozopsis similis was reinvestigated using GT335, an antibody binding to polyglutamylated tubulins, and antibodies directed to p210, a component of the flagellar transition region which represents the distal border of the basal body. p210 was also detected in small spots at the base of each basal body which increased in size prior to mitosis. The presence of p210 on one of the microtubular flagellar roots suggested a transport of basal body material along these tracks. Immunogold electron microscopy confirmed the presence of p210 in the probasal bodies. Further, small probasal bodies are apparently connected to the mature basal bodies by centrin fibers as observed after artificially induced basal body separation in Xenopus egg extract. While basal bodies grew, most of the p210 remained at the tip of elongating basal bodies, but two or four additional spots were observed in distinct patterns near the base of the basal bodies. In cytokinesis, basal body pairs separated and p210 was observed in a strong signal at the tip and a weaker one in the vicinity of the proximal end of each basal body. We interpret the data as indicating that a new p210-containing structure forms near the proximal end of the basal bodies during basal body elongation, representing the precursor of the next generation of basal bodies. Thus, basal bodies appear to seed the succeeding generation already during their own development, a mechanism which could ensure the correct number and position of basal bodies.     

The basal body-root complex ofChlamydomonas reinhardtii during mitosis

Summary The results of this work clarify several structural and temporal aspects of biogenesis of the basal body-root complex inChlamydomonas reinhardtii. The two phases of basal body development (probasal body assembly and conversion of probasal body into mature basal body) occur at identical mitotic stages in successive mitoses during multiple fission, which indicates a tight coupling between basal body development and the mitotic cycle. The two steps of basal body development are separated from one another in time,i.e. immature probasal bodies originate during an interval lasting ca. 5 min between mid-metaphase and early telophase, but mature after a quasi-dormant period only during early prophase of the next mitotic round. The duration of the dormant period depends on the interval between two mitoses: during synchronized vegetative growth there is an interval of ca. 20 h (interphase growth) between two rounds of multiple fissions, but only a maximum interval of 1.5 h between the successive mitoses of one round of multiple fissions.The microtubular root system, which is bisected at the same time as the basal body apparatus in a plane perpendicular to the distal connecting fiber during prophase, and whose roots seem to be reduced in length, starts duplication at early metaphase with the successive origin of two short bud-like partner roots just opposite the remnants. These initial roots elongate during subsequent phases by unilateral and radial growth from the basal bodies and along the cell's periphery, but exactly where they terminate is not known. The two-stranded roots opposite each other appear to be again connected as early as anaphase.The striation pattern of the distal connecting fiber is lost during early prophase thus indicating a partial breakdown of the fiber.Dedicated to Prof. Dr. C.-G. Arnold (Erlangen) on the occasion of his 60th birthday.     

Basal Body Components Exhibit Differential Protein Dynamics during Nascent Basal Body Assembly

Basal bodies organize cilia that are responsible for both mechanical beating and sensation. Nascent basal body assembly follows a series of well characterized morphological events; however, the proteins and their assembly dynamics for new basal body formation and function are not well understood. High-resolution light and electron microscopy studies were performed in Tetrahymena thermophila to determine how proteins assemble into the structure. We identify unique dynamics at basal bodies for each of the four proteins analyzed (α-tubulin, Spag6, centrin, and Sas6a). α-Tubulin incorporates only during new basal body assembly, Spag6 continuously exchanges at basal bodies, and centrin and Sas6a exhibit both of these patterns. Centrin loads and exchanges at the basal body distal end and stably incorporates during new basal body assembly at the nascent site of assembly and the microtubule cylinder. Conversely, both dynamic and stable populations of Sas6a are found only at a single site, the cartwheel. The bimodal dynamics found for centrin and Sas6a reveal unique protein assembly mechanisms at basal bodies that may reflect novel functions for these important basal body and centriolar proteins.     

Centrin2 regulates CP110 removal in primary cilium formation

Primary cilia are antenna-like sensory microtubule structures that extend from basal bodies, plasma membrane–docked mother centrioles. Cellular quiescence potentiates ciliogenesis, but the regulation of basal body formation is not fully understood. We used reverse genetics to test the role of the small calcium-binding protein, centrin2, in ciliogenesis. Primary cilia arise in most cell types but have not been described in lymphocytes. We show here that serum starvation of transformed, cultured B and T cells caused primary ciliogenesis. Efficient ciliogenesis in chicken DT40 B lymphocytes required centrin2. We disrupted CETN2 in human retinal pigmented epithelial cells, and despite having intact centrioles, they were unable to make cilia upon serum starvation, showing abnormal localization of distal appendage proteins and failing to remove the ciliation inhibitor CP110. Knockdown of CP110 rescued ciliation in CETN2-deficient cells. Thus, centrin2 regulates primary ciliogenesis through controlling CP110 levels.     

Fine Structure of Microgametocytes and Macrogametes of Eimeria nieschulzi

SYNOPSIS. An electron microscope study of microgametocytes and macrogametes of Eimeria nieschulzi Dieben, 1924 revealed that they lie within vacuoles bounded by a host unit membrane. The vacuole surrounding the microgametocyte contains granular material. The vacuole around the macrogamete is narrower and contains vesicles and membranes. Micropores were seen on the surface of the plasma membrane of microgametocytes and macrogametes. Microtubules were seen in macrogametes. Young microgametocytes and macrogametes have a similar cytoplasmic matrix, mitochondria and nuclei. Glycogen granules apparently develop around vacuoles in both microgametocytes and macrogametes. Glycogen granules were also seen along the margins of parallel bundles of fibers in microgametocytes. As nuclei of the microgametocyte divide, they move to the periphery of the parasite. Three basal bodies, each with 9 fibers in triplet form, develop in association with each nucleus. Microgametes have 2 free flagella and a central short, attached flagellum. Basal granules lie along the outer fibers of the central flagellum. Each microgamete has an elongate mitochondrion in close contact with the nucleus. In macrogametes wall-forming bodies develop in lacunae in the cytoplasm. Smaller dark bodies with areas of low density were also seen. Wall-forming bodies and dark bodies move to the periphery of mature macrogametes.     

Ofd1 Controls Dorso-Ventral Patterning and Axoneme Elongation during Embryonic Brain Development

Oral-facial-digital type I syndrome (OFDI) is a human X-linked dominant-male-lethal developmental disorder caused by mutations in the OFD1 gene. Similar to other inherited disorders associated to ciliary dysfunction OFD type I patients display neurological abnormalities. We characterized the neuronal phenotype that results from Ofd1 inactivation in early phases of mouse embryonic development and at post-natal stages. We determined that Ofd1 plays a crucial role in forebrain development, and in particular, in the control of dorso-ventral patterning and early corticogenesis. We observed abnormal activation of Sonic hedgehog (Shh), a major pathway modulating brain development. Ultrastructural studies demonstrated that early Ofd1 inactivation results in the absence of ciliary axonemes despite the presence of mature basal bodies that are correctly orientated and docked. Ofd1 inducible-mediated inactivation at birth does not affect ciliogenesis in the cortex, suggesting a developmental stage-dependent role for a basal body protein in ciliogenesis. Moreover, we showed defects in cytoskeletal organization and apical-basal polarity in Ofd1 mutant embryos, most likely due to lack of ciliary axonemes. Thus, the present study identifies Ofd1 as a developmental disease gene that is critical for forebrain development and ciliogenesis in embryonic life, and indicates that Ofd1 functions after docking and before elaboration of the axoneme in vivo.     

The UNI1 and UNI2 Genes Function in the Transition of Triplet to Doublet Microtubules between the Centriole and Cilium in Chlamydomonas

One fundamental role of the centriole in eukaryotic cells is to nucleate the growth of cilia. The unicellular alga Chlamydomonas reinhardtii provides a simple genetic system to study the role of the centriole in ciliogenesis. Wild-type cells are biflagellate, but “uni” mutations result in failure of some centrioles (basal bodies) to assemble cilia (flagella). Serial transverse sections through basal bodies in uni1 and uni2 single and double mutant cells revealed a previously undescribed defect in the transition of triplet microtubules to doublet microtubules, a defect correlated with failure to assemble flagella. Phosphorylation of the Uni2 protein is reduced in uni1 mutant cells. Immunogold electron microscopy showed that the Uni2 protein localizes at the distal end of the basal body where microtubule transition occurs. These results provide the first mechanistic insights into the function of UNI1 and UNI2 genes in the pathway mediating assembly of doublet microtubules in the axoneme from triplet microtubules in the basal body template.     

A Novel 95-kD Protein Is Located in a Linker between Cytoplasmic Microtubules and Basal Bodies in a Green Flagellate and Forms Striated Filaments In Vitro

The flagellar basal apparatus comprises the basal bodies and the attached fibrous structures, which together form the organizing center for the cytoskeleton in many flagellated cells. Basal apparatus were isolated from the naked green flagellate Spermatozopsis similis and shown to be composed of several dozens of different polypeptides including a protein band of 95 kD. Screening of a cDNA library of S. similis with a polyclonal antibody raised against the 95-kD band resulted in a full-length clone coding for a novel protein of 834 amino acids (90.3 kD). Sequence analysis identified nonhelical NH₂- and COOH-terminal domains flanking a central domain of ~650 residues, which was predicted to form a series of coiled-coils interrupted by short spacer segments. Immunogold labeling using a polyclonal antibody raised against the bacterially expressed 95-kD protein exclusively decorated the striated, wedge-shaped fibers, termed sinister fibers (sf-fibers), attached to the basal bodies of S. similis. Striated fibers with a periodicity of 98 nm were assembled in vitro from the purified protein expressed from the cloned cDNA indicating that the 95-kD protein could be a major component of the sf-fibers. This structure interconnects specific triplets of the basal bodies with the microtubular bundles that emerge from the basal apparatus. The sf-fibers and similar structures, e.g., basal feet or satellites, described in various eukaryotes including vertebrates, may be representative for cytoskeletal elements involved in positioning of basal bodies/centrioles with respect to cytoskeletal microtubules and vice versa.     

Determination of ciliary polarity precedes differentiation in the epithelial cells of quail oviduct.

In quail oviduct epithelium, as in all metazoan and protozoan ciliated cells, cilia beat in a coordinated cycle. They are arranged in a polarized pattern oriented according to the anteroposterior axis of the oviduct and are most likely responsible for transport of the ovum and egg white proteins from the infundibulum toward the uterus. Orientation of ciliary beating is related to that of the basal bodies, indicated by the location of the lateral basal foot, which points in the direction of the active stroke of ciliary beating. This arrangement of the ciliary cortex occurs as the ultimate step in ciliogenesis and following the oviduct development. Cilia first develop in a random orientation and reorient later, simultaneously with the development of the cortical cytoskeleton. In order to know when the final orientation of basal bodies and cilia is determined in the course of oviduct development, microsurgical reversal of a segment of the immature oviduct was performed. Then, after hormone-induced development and ciliogenesis, ciliary orientation was examined in the inverted segment and in normal parts of the ciliated epithelium. In the inverted segment, orientation was reversed, as shown by a video recording of the direction of effective flow produced by beating cilia, by the three-dimensional bending forms of cilia immobilized during the beating cycle and screened by scanning electron microscopy, and by the position of basal body appendages as seen in thin sections by transmission electron microscopy. These results demonstrate that basal body and ciliary orientation are irreversibly determined prior to development by an endogenous signal present early in the cells of the immature oviduct, transmitted to daughter cells during the proliferative phase and expressed at the end of ciliogenesis.     

Ultrastructural Localization of Acid Phosphatase in Feeding Tokophrya infusionum*

A combined cytochemical and electronmicroscopic study of feeding Tokophrya revealed that it has 2 sources of acid phosphatase. One is from the prey, Tetrahymena, supplying newly formed food vacuoles with large amounts of enzyme. The other source is in Tokophrya itself, the enzyme being found in small vesicles, small dense elongate bodies surrounded by a membrane, or in residue vacuoles. It seems that the 2 former small structures contain insignificantly small amounts of phosphatase; however, large deposits of lead phosphate are present in residue vacuoles, former food vacuoles. Since Tokophrya has no cytopyge these vacuoles are not excreted. On the contrary, when feeding is resumed, they merge with food vacuoles, presumably supplying them with acid phosphatase. Whether this enzyme ultimately is derived from the prey Tetrahymena and persists undegraded in the residue vacuoles, or whether it is synthesized by Tokophrya cannot be determined from present work.     

THE CYTOSKELETON OF THE NAKED GREEN FLAGELLATE SPERMATOZOPSZS SIMILIS (CHLOROPHYTA): ANALYSIS OF ISOLATED BASAL APPARATUSES IN THE PARALLEL CONFIGURATION1,2

The biflagellate green alga Spermatozopsis similis is demonstrated to be a model organism for the biochemical and functional analysis of the basal apparatus. Basal apparatuses were isolated in the presence of 10⁻⁶ M Ca²⁺, which induces the reorientation of the basal bodies into the parallel state. Serial thin sectioning of enriched basal apparatuses stained with tannic acid reveals several novel details of the structure of the basal bodies, the distal connecting fiber, and the striated microtubule-associated fibers. We observed a pronounced difference in size of a striated fiber connecting the basal bodies to the five-stranded microtubular roots depending on its association with the developmentally older or younger basal body. Instead of a proximal connecting fiber, the proximal end of each basal body is associated with a striated triangular plate; these plates appear to serve as spacers for the basal bodies in the parallel and antiparallel configurations. We suggest that the plates play a role in maintaining basal body orientation during forward and backward swimming. The results are summarized in representative drawings of the basal apparatus.     

Cytochalasin D inhibits basal body migration and ciliary elongation in quail oviduct epithelium

Summary The effects of cytochalasin D (CD) were studied by scanning (SEM) and transmission (TEM) electron-microscopic examination at different stages of ciliary differentiation in epithelial cells of quail oviduct. Immature quails were prestimulated by estradiol benzoate injections to induce ciliogenesis in the undifferentiated oviduct. After 24 h of CD culture, SEM study revealed inhibition of ciliogenesis and dilation of the apex of non-ciliated cells. TEM study showed that 2 h of CD treatment produced dilation of lateral intercellular spaces, after 6 h of treatment, this resulted in intracellular macrovacuolation. Vacuoles were surrounded by aggregates of dense felt-like material. CD also induced the disappearance of microvilli, and rounding of the apical surface of undifferentiated cells and those blocked in ciliogenesis. Centriologenesis was not inhibited by CD; basal bodies assembled in generative complexes in the supranuclear region after 24 h of treatment. However, the migration of mature basal bodies towards the apical surface was impaired. Instead, they anchored onto the membrane of intracellular vacuoles; growth of cilia was induced in the vacuole lumen. Cilium elongation was disturbed, giving abnormally short cilia with a dilated tip; microtubules failed to organize correctly.